Cells were grown in Balch medium III at 35 °C with shaking (Balch et al., 1979; Kalmokoff et al., 1988). An inframe deletion mutant in flaK derived from M. maripaludis Mm900 was described previously (Ng et al., 2009). These cells are nonflagellated, but piliated and complementation of flaK in trans restores flagellation. Using the flaK mutant as a starting host, the subsequent deletion of the prepilin peptidase eppA was accomplished using the technique of Moore & Leigh (2005). An approximately 1-kb region upstream

of eppA was amplified using the primers P1: 5′-CGCGGATCCCATTTCTATCAATTTTCCAC and P2: 5′-TTGGCGCGCCGGGGAATTATTCGCTCTTTGATAT. Primers P3: 5′-TTGGCGCGCCGGCGTTATAAATTATCTGGTGGGA and P4: 5′-CGCGGATCCCGTTTGACTGTTTGAACAGC Kinase Inhibitor Library were used to amplify approximately 1 kb downstream of the gene. Both P2 and P3 primers had AscI sites incorporated into them (underlined in primer), allowing for Selleckchem CH5424802 AscI cleavage of the two PCR products, followed by ligation and PCR amplification with primers P1 and P4 to generate an approximately 2-kb fragment that contained an inframe deletion version of eppA. Using the BamHI sites

incorporated into primers P1 and P4 (underlined), this piece was cloned into pCRPrtNeo and transformed into the existing M. maripaludis flaK deletion strain with transformants screened for the eppA deletion by PCR using primers P5: 5′-CTGGAGCTGTATGAAATGCAACTGG and P6: 5′-CCTGCATTATCCCAGGTCATCC, which amplify across the deleted region. Similarly, the same plasmid was transformed into the Mm900 strain and transformants screened for the check eppA deletion leading to mutants that were wild type for flaK, but deleted only for eppA. Wild-type and mutants cells were grown for 18 h at 35 °C before ethanol-sterilized substrates to be tested for attachment were added. Incubation continued at 35 °C with gentle agitation for a further 24 h. Tested substrates

included various grids [200 or 400 mesh uncoated gold, nickel (Agar Scientific, Essex, UK) and molybdenum grids (Gilder Grids, Grantham, UK)], mica, silicon wafer chips (Agar Scientific) and glass. To examine whether surface contact influenced the production of pili, the flaK deletion mutant was grown on Balch medium III plates (with 1.5% w/v Noble agar) for 4 days. Colonies were removed, resuspended in medium and briefly centrifuged. The pellet was gently resuspended in 2% glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.2–7.4, containing 2% w/v NaCl for 30 min and examined by transmission electron microscopy (TEM), as described below. Wild-type and mutant cells were examined by TEM to identify the presence of surface appendages. Cells were fixed with 2% glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.2–7.